This invention relates to film based addressable programmable electronic matrix articles and methods for manufacture and use of the same. The articles are manufactured on a flexible film, and are suited for use with patterned polymer films tailored to selectively bind or react with various target species, including biologically active molecules.
One of the most important activities of modern medical and biochemical fields is conducting medical diagnostic assays, such as cell culture assays, immunoassays, DNA hybridization assays, robot assisted sample handling processes, and microfluid sample processing. These activities permit safe and effective medical diagnoses, as well as thorough and accurate biochemical investigations and research.
Automated microbial culturing systems have been developed in recent years to test for a variety of diseases in a clinical laboratory setting. These systems often include culture tubes containing samples, selective growth media, and a fluorescent indicator that responds to the growth of the microorganisms. The tubes are continually processed by an optical reader that measures changes in fluorescent properties of the sample in order to detect such microbes as tuberculosis or antibiotic resistant Staph. aureus. Unfortunately, such systems can take days to culture a sufficient quantity of the microbes necessary for identification with standard test methods.
Biocards. such as those described in U.S. Pat. No. 5,609,828, have also been developed to carry out multiple assays from a single sample extracted from blood, fluids, or other tissue of a patient. These samples are usually examined using spectroscopic or other automated analysis techniques. Biocards are typically molded in plastic and are designed to receive a liquid sample into a series of small sample wells formed in the card. Each sample well normally contains a different set of dried reagent (selective growth nutrients and indicator dies) for identifying different biological agents within the sample. During analysis, the sample enters an intake port, collects in an intake reservoir, and travels along distribution channels to the sample wells. Each sample well also typically includes a bubble trap designed to trap gases formed by growing microorganism colonies. The reagents within the sample wells dissolve when the fluid sample is introduced. After incubation of the sample in the sample wells, a card reader performs automated spectroscopic or fluorescence analysis on each well. Although analysis with biocards can be successful, analysis times are quite long unless the microorganisms are first cultured to increase their number. In addition, closely related strains of microorganisms are hard to differentiate by these methods.
Efforts have been made to develop assay techniques for the analysis of nucleic acids and proteins that shorten the delay associated with culture techniques, increase the specificity of the assays, and provide means for detecting new diseases. One such effort has been the development of DNA amplification technologies that provide a means to produce hundreds of millions of copies of a selected DNA target in less than one hour. Microorganisms of interest are first lysed to release their DNA material. The DNA material is isolated and then treated with reagents to perform an amplification of an oligonucleotide sequence specific to the microorganism of interest. While polymerase chain reaction (PCR) is the most well known of these amplification methods, it requires temperature cycling and continued reagent additions. Other methods, such as a strand displacement amplification approach developed at Becton Dickinson of Franklin Lakes, N.J., can be performed in a single sample well in 15 minutes at constant temperature. Such amplification methods help to overcome problems related to complexity and sensitivity in genomic DNA analysis.
Once the DNA target has been xe2x80x9camplifiedxe2x80x9d by reproduction to produce numerous amplicons, complementary oligonucleotide probes can be used to capture the DNA amplicons. These probes selectively retain the DNA amplicon, allowing them to be isolated and identified. However, these probes have traditionally relied upon diffusion controlled processes to capture the DNA amplicons. Diffusion can take hours to complete, and is a significant hurdle to rapid identification of the DNA target. Although such diffusion methods are significantly quicker than prior cell culture techniques, they are still relatively slow compared to the rapid rate of DNA amplification.
An alternative to DNA amplification, known as the Southern Blot, involves cleaving the DNA with restrictive enzymes, separating the DNA fragments on an electrophoresis gel, blotting to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity of the crude DNA sample, thereby helping to improve the hybridization specificity and sensitivity. However, the total number of targets generated in a Southern Blot is far less than the number of amplicons generated by DNA amplification methods. In addition, the electrophoretic separation can take hours to complete.
Recently, efforts have been made to hasten the separation and capture of DNA amplicons. Researchers have developed micro-electrode arrays that speed up the capture process by using free-field electrophoresis to concentrate and purify target DNA on the individual probes of the array. These arrays create a charged electrical field that isolates the charged DNA amplicons at one or more probes on the array. This type of microelectronic array, known as an xe2x80x9caddressable programmable electronic matrixxe2x80x9d (xe2x80x9cAPEXxe2x80x9d), can reduce the time to perform the capture process from hours to minutes. A number of patents describe silicon based chips having APEX arrays. For example, U. S. Pat. Nos. 5,653,939 and 5,632,957 teach the manufacture-of rigid silicon APEX arrays using a lithographic process. Although these silicon APEX arrays permit enhanced capture rates, they are relatively expensive to produce. Also, they cannot be easily reused without being cleaned and repatterned with DNA probes in a manufacturing environment. Even though they are relatively expensive, the silicon APEX arrays are typically used once and then discarded.
Thus, existing APEX arrays have improved the speed of performing DNA identification tests, but have failed to address the need for cost-effective, mass manufacturable APEX assay systems. In view of the significant expense associated with existing APEX chip systems, a need exists for an APEX chip that provides the increased speed of a programmable microelectronic matrix, but can be done for less expense than existing silicon-based chips.
The present invention is directed to a film based microelectrode array device adapted for processing of chemical, biological, or particulate materials at electronically addressed micro-locations. Uses for the device include chemical- and molecular biological-type analyses, including nucleic acid hybridization reactions, antibody/antigen reactions, various cell sorting operations, and synthesis reactions including DNA amplifications and various free-field electrophoresis manipulations.
In specific implementations, the device includes a flexible polymeric substrate having a first or upper surface and a second or lower surface. A plurality of microlocations interrupts the first surface, and each of these microlocations preferably includes an electrode disposed on the first or second surface of the flexible substrate. Circuit traces connect the electrodes to larger contact pads also disposed on the first or second surface of the flexible substrate. A hydrophilic permeation matrix and optionally a biologically receptive polymer is positioned on the first surface of the flexible substrate and is capable of supporting electrical contact between the electrode and a fluid sample placed in contact with the first surface of the flexible substrate. Flexible polymeric substrates of the present invention are particularly well suited for the manufacture of APEX chips and enable the manufacture of novel APEX biocards, and APEX spools.
In certain implementations of the invention, the flexible polymeric substrate is formed of polyimide. The flexible polymeric substrate may be completely, substantially, or partially formed of the polyimide, and may be combined with other materials. It will be appreciated that the invention is also directed to any flexible substrate that permits the formation of a first surface and a second surface, with electrodes interrupting the first surface. The substrate and electrodes should also permit the retention of a hydrophilic permeation matrix, such as a derivitizable gel, in a manner such that programmable free-field electrophoresis processes can be conducted using the substrate and electrodes. The hydrophilic permeation matrix may include a biologically receptive gel, or may have inherent biological receptive properties.
In one implementation, the plurality of microlocations interrupting the first surface includes vias that extend from the first surface into the polymeric substrate. The vias provide a location for the formation of the electrodes. The electrodes are formed, for example, by securing a conducting metallic layer on the second or lower surface of the flexible substrate and then selectively removing the polymeric substrate above the conducting metallic layer. The polymeric substrate is selectively removed to expose the conducting metallic layer, thereby forming electrodes. Removal of the substrate is performed by chemical etching methods, plasma removal methods, laser removal methods, or other suitable methods.
In other examples, the electrodes are formed by depositing a metallic layer on the first or upper surface of the flexible polymeric sheet. After the metallic layer is deposited, a non-conductive masking layer is applied over the metallic layer. This masking layer is selectively removed by an appropriate methodology, such as photo-lithography, in order to expose portions of the metallic layer to form a plurality of exposed electrodes.
It will also be appreciated that in certain implementations of the invention the electrodes formed by exposure of the metal layer may subsequently be enlarged by deposition of additional metal on the electrode. The deposition of additional metal is accomplished, for example, by electroplating the electrodes to selectively thicken them. Alternatively, small metallic pieces may be mechanically inserted into each via above each electrode, and these metallic pieces can be subsequently fused by heat or pressure in order to thicken the electrode at the microlocation. Such thickening of the electrode can enhance performance of the APEX by allowing tailoring of the contact surface, e.g., by selection of electrode metals such as gold. Also, the additional metal can enhance performance by effectively securing the metal layer to the substrate by fusing the electrode within the via.
The electrodes formed by exposure to the metal layer may be connected by metal traces to much larger contact pads located elsewhere on the first or second surface of the flexible polymeric substrate. When the electrodes are connected by vias to the circuit traces and contact pads printed on the second surface of the substrate, the first surface bearing the exposed electrodes can be directly laminated to a fluid handling architecture that directs the fluid sample to the electrode array. The contact pads on the second surface can be designed to extend to the edge of the device and mate directly with a voltage control unit by sliding the chip into a mated connector. This design overcomes arduous wire bonding processes of prior art APEX chips and overcomes the need to encapsulate the lead wires in a protective material, since they are shielded by the flexible polymeric substrate from exposure to the fluid sample.
The electrodes may be recessed within the vias in the flexible polymeric substrate and routed through conducting traces on the second surface of the flexible polymer substrate. Such a one-piece construction comprises a microwell above the electrode and provides protection for the circuit traces without the need for an additional protective layer. The microwell can serve as a reservoir for a sample or it can be partially or completely filled with a hydrophilic polymer to introduce a permeation layer or a biologically receptive gel above the electrode. In this manner the improved APEX device of the present invention overcomes the difficulties in mating a circuit board to a microwell array, as described in U.S. Pat. No. 5,605,662.
A hydrophilic permeation matrix on the top surface of the electrodes permits free-field electrophoresis of samples placed on the top surface of the APEX circuit while, at the same time, impeding the diffusion of large biomolecules, biologically receptive molecules, biologically reactive molecules, reagents or products through the matrix to the surface of the electrode. This matrix may be biologically receptive and used to attach biomolecules to the electrode. In certain implementations, a separate biologically receptive gel may be chemically or physically adhered to the permeation matrix or the electrode itself to support the attachment of biomolecules. The biologically receptive gel or permeation matrix may include an azlactone-functional monomer. As used in the present invention, xe2x80x9cazlactone-functional monomerxe2x80x9d means a monomer whose structure includes an azlactone moiety that optionally has been bound to a biomolecule by a ring opening reaction of the azlactone to form, e.g., an amide bond. The gel is preferably swellable, thereby providing an increased concentration of biologically receptive molecules per unit area when used as a coating. The biologically receptive gel may be configured and arranged such that it may be patterned to a high resolution. This gel may be cured by actinic radiation, and the cured composition may be capable of reacting with selective biomolecules to immobilize the biomolecules immediately above the micro-electrodes.
An azlactone-functional gel or permeation matrix can be employed and localized to the region just above the electrodes and within the confines of vias, in a specific embodiment of the invention. In this embodiment, some or all of the azlactone groups over a particular electrode can be reacted, through simple addition reactions, with selected amine- or thio-terminated biomolecules to immobilize these biomolecules above the electrode, thus forming a biologically receptive gel. These selected biomolecules may be oligonucleotide or antibody probes, enzymes such as polymerase, or other biomolecules useful for analysis. One advantage of the azlactone based APEX films of the present invention is that the azlactone and the biomolecules can be anchored to the microlocations of the film by simple web coating, inkjet printing, or thermal imaging technologies, without the need for reagent additions or product removal, thereby greatly simplifying their manufacture relative to prior art APEX chips.
As used in the present invention, xe2x80x9chydrophilic permeation matrixxe2x80x9d is a polymeric material capable of swelling in water, such as by adsorption or chemical interaction, and refers to the matrix material either before or after swelling in water. xe2x80x9cPhotocrosslinkerxe2x80x9d means a chemical species that is capable of binding two or more polymer molecules in response to the application of electromagnetic radiation. The photocrosslinker is capable of attaching to the polymer molecules at a site other than the end of a growing polymer chain. xe2x80x9cCopolymeric crosslinkerxe2x80x9d means a chemical species that is capable of binding two or more polymer molecules, and that attaches to a polymer at the end of a growing polymer chain.
As used herein, xe2x80x9cbiologically activexe2x80x9d includes biochemically, immunochemically, physiologically. or pharmaceutically active; and xe2x80x9cbiologically active moleculexe2x80x9d and xe2x80x9cbiomoleculexe2x80x9d are used interchangeably and include antibodies, antigens, enzymes, cofactors, inhibitors, hormones, receptors, coagulation factors, amino acids, histories, vitamins, drugs, cell surface markers, carbohydrates, proteins and polypeptides, DNA (including DNA oligonucleotides), RNA (including RNA oligonucleotides), and derivatives of the foregoing. xe2x80x9cSubstitutedxe2x80x9d means substituted by conventional substituents which do not interfere with the desired product, e.g., substituents can be alkyl, alkoxy, aryl, phenyl, halo (F, Cl, Br, I), cyano, nitro, etc.
The invention provides a flexible polymeric substrate that can be continuously produced on a commercial scale in the convenient form of a roll-good that can be readily stored and handled. The finished roll-good can be used directly after application of the hydrophilic matrix or biologically active gel to perform electrophoresis assisted processing of chemical, biological, or particulate materials at electronically addressed micro-locations. For example, the flexible polymeric substrate can be used on a spool or roll-good in a continuous reel-to-reel process in which a plurality of addressable programmable electrode matrices are sequentially supplied, used, and taken up. Alternatively, the roll-good can be cut into sections containing a plurality of APEX arrays for incorporation into biocards. Also, alternatively, the roll-good may be cut into separate APEX units for individual use. It will be appreciated that the flexible polymeric substrate containing the APEX unit or units can further include or be combined with other microelectronic, microoptical, microstructural, and/or micromechanical elements. These microelements may be incorporated into multilayer articles.
The invention is further directed to a multi-sample APEX processing spool and system in which the first surface of a roll of the APEX film may be laminated to a flexible plastic fluid handling architecture, said fluid handling architecture designed to direct 2-10,000 independent biological samples to the corresponding number of independently addressable APEX arrays on the APEX film for processing. The spool can be advanced through a machine that infects a sample into a subset of the APEX arrays for processing. Once the samples are assayed, the spool can be advanced, exposing additional APEX arrays. A voltage control unit simultaneously can provide processing currents or voltages to each of the APEX arrays on the spool at any one time. A detection system can provide optical, electrical, or mechanical signals in response to the biological events at the individual electrodes of each APEX array. Thus, continuous automated processing, whether sequentially or simultaneously, of thousands of samples can be carried out using inexpensive, disposable arrays.
The invention is further directed to a multi-sample APEX biocard and system in which a sheet of APEX film may be laminated to a semi-rigid glass or plastic fluid handling architecture, said fluid handling architecture designed to direct numerous independent biological samples to the corresponding number of independently addressable APEX arrays on the APEX film for processing, preferably between 2 and 200 samples. The fluid handling architecture can be anything from simple barriers between APEX arrays, including open wells into which sample is injected when the cassette is horizontal, to closed channel structures into which a sample is injected with a syringe or pump. A machine can be adapted to accept and operate the APEX cassette. The machine may comprise a sample injection unit to provide numerous (preferably from 2 to 200) independent biological samples onto the biocard via ports in the fluid handling architecture; a voltage control unit that can simultaneously provide processing currents or voltages to each of the APEX arrays; and a detection system that can provide optical, electrical, or mechanical signals in response to biological events at the individual electrodes of each APEX array.
The invention is further directed to a method of performing molecular biological processes, the method including providing an electronic device containing a flexible polymeric substrate having a first surface and a second surface. The electronic device may comprise an array of electrodes disposed on the first or second surfaces of the flexible substrate and exposed toward the first surface. A hydrophilic permeation matrix and biologically receptive polymer having a covalently anchored biological molecule can be positioned on the first surface of the flexible substrate such that they are in contact with the array of electrodes, after which an electrical force can be applied to the electrodes so as to effect electrophorcsis-assisted processing of the biological sample. By similar means, electrophoretic processing of chemical or particulate matter also can be envisioned.
In specific implementations of the method of the invention, the electronic device may include a plurality of electrodes. The charge potential of the electrodes preferably can be individually controllable. Alternatively, the charge potential of the electrodes can be controlled together as one unit, or as a group of sub-units, each sub-unit having a plurality of electrodes electronically coupled to one another. During operation, the charge potential of the electrodes optionally can be altered in order to modify the electrical field of the electronic device. Such alterations to the electrical field can be used, for example, to first attract all the charged biologically active molecules to the biologically receptive gel, and then to subsequently repel the biologically active molecules that are not retained by the receptive gel. These efforts can result in accumulation of desired biomolecules and removal of undesired molecules from the positions of the electrodes based on specific biomolecular recognition by anti-body or oligonucleotide probes associated with the individual micro-electrodes.
Other features and advantages of the invention will be apparent from the following detailed description of the invention and the claims. The above summary of principles of the disclosure is not intended to describe each illustrated embodiment or every implementation of the present disclosure. The figures and the detailed description that follow more particularly exemplify certain embodiments utilizing the principles disclosed herein.